Methods for the detection and quantitation of hydroethidine (HE) and its oxidation products by HPLC analysis are described. Synthetic methods for preparation of authentic standards (2-hydroxyethidium and diethidium) are provided. Potential applications of the HPLC methods to chemical and biological systems are discussed. Specific examples of chromatograms obtained using UV-Vis absorption, fluorescence, electrochemical, and mass spectrometry detectors are provided. The development of a dual electrochemical and fluorescence detection methodology and its applications are described. The HPLC-based method enables analyses of HE and its oxidation products such as ethidium and the dimeric products of HE. The ramifications of HPLC measurement of HE and its oxidation products in the detection and quantitation of 2-hydroxyethidium, the diagnostic marker product of superoxide and HE, in the intracellular milieu are discussed. Similarly, mitochondria-targeted HE conjugated to a triphenylphosphonium group (Mito-HE or Mito-SOX) also forms oxidation products (dimers of Mito-HE and Mito-E+) that can affect the detection and quantitation of 2-hydroxy-mito-ethidium, the diagnostic marker product of Mito-HE and superoxide in mitochondria.